Abstract Defects in apoptotic capability that evolve during tumorigenesis promote tumor growth, provide survival advantage, and confound treatment. Such defects have been a major hurdle in the development of TRAIL therapy. Targeting TRAIL-Rs with either recombinant TRAIL or agonistic DR4 or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells to TRAIL over normal cells. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our preliminary studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained substantial evidence that inhibition of such a TRAIL-mediated autophagic response initiates an effective apoptotic cascade. We propose to investigate a novel concept that divergent mechanisms of resistance to TRAIL- mediated apoptosis involve the induction of protective autophagy, and thus can be reversed by targeting for inhibition specific components of the autophagic process. We hypothesize that inhibition of autophagy can be utilized for TRAIL therapy since it reverses certain mechanisms of TRAIL resistance in apoptosis- defective tumor cells. We further hypothesize that essential components of the autophagosome formation process, particularly Beclin-1/Atg6, UVRAG, Vps34 and Atg7, are involved in a constant crosstalk between autophagy and apoptosis through their dual function of mediating autophagy and inhibiting apoptosis. Thus, the knockdown of such proteins would not only block autophagy, but would also initiate apoptotic events in a manner that depends on the antiapoptotic function of the targeted Atg protein. Although the proposed studies focus on mechanisms of resistance to TRAIL therapy, they are highly relevant to a broader goal of eradicating multidrug-resistant cancer cells, as they target a major stumbling block in numerous cytotoxic regimens, i.e., defects in apoptotic capability that evolve during tumorigenesis. The proposed studies are designed to elucidate molecular mechanisms involved in the mutual inhibition between autophagy and apoptosis. A better understanding of the molecular basis for the cross regulation between autophagy and apoptosis will help determine if modulation of autophagy can be utilized for cancer therapy. PUBLIC HEALTH RELEVANCE: Our preliminary results suggest that numerous cellular mechanisms within tumor cells that commonly interfere with the efficacy of the anticancer drug, TRAIL, involve the activation of a cytoprotective response. We also obtained evidence that inhibition of such a cellular protective response yields a productive cell death. The goal of the current application is to elucidate the molecular mechanisms that underlie the reversal of tumor cell resistance to TRAIL therapy.